An ectopic enhancer restores CFTR expression through de novo chromatin looping [4C-seq]
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ABSTRACT:
PROVIDER: PRJNA841376 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR19350321.fastq.gz | Fastqsanger.gz | |||
| SRR19350322.fastq.gz | Fastqsanger.gz | |||
| SRR19350323.fastq.gz | Fastqsanger.gz | |||
| SRR19350324.fastq.gz | Fastqsanger.gz | |||
| SRR19350325.fastq.gz | Fastqsanger.gz |
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Project description:Transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is regulated by a set of ubiquitous and cell-type selective cis-regulatory elements (CREs). These elements include extragenic and intronic enhancers that bind cell-type specific transcription factors, as well as architectural protein bound structural elements. We previously reported that loss of the airway-selective -35kb enhancer in 16HBE14o- airway epithelial cells disrupts normal enhancer-promoter looping at the CFTR locus and nearly abolishes its expression. Here we expand on previous work, and explore the effect of relocation of the 500bp -35kb enhancer core to an ectopic site in intron 1 of CFTR on gene expression and chromatin dynamics. We find that although genomic relocation of this element is not able to fully restore CFTR expression to wild-type levels, the -35kb sequence is able establish a de novo enhancer signature at chromatin at the site of insertion. Additionally, the relocated -35kb enhancer is able to form chromatin contacts with known CFTR CREs. These studies reveal important direct chromatin effects, as well as limitations, of CFTR enhancers, and provide important considerations when targeting the CFTR locus for novel therapeutic gene editing.
2022-12-13 | GSE203559 | GEO
Project description:An ectopic enhancer restores CFTR expression through de novo chromatin looping.
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Project description:An ectopic enhancer restores CFTR expression through de novo chromatin looping [ATAC-seq]
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Project description:Transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is regulated by a set of ubiquitous and cell-type selective cis-regulatory elements (CREs). These elements include extragenic and intronic enhancers that bind cell-type specific transcription factors, as well as architectural protein bound structural elements. We previously reported that loss of the airway-selective -35kb enhancer in 16HBE14o- airway epithelial cells disrupts normal enhancer-promoter looping at the CFTR locus and nearly abolishes its expression. Here we expand on previous work, and explore the effect of relocation of the 500bp -35kb enhancer core to an ectopic site in intron 1 of CFTR on gene expression and chromatin dynamics. We find that although genomic relocation of this element is not able to fully restore CFTR expression to wild-type levels, the -35kb sequence is able establish a de novo enhancer signature at chromatin at the site of insertion. Additionally, the relocated -35kb enhancer is able to form chromatin contacts with known CFTR CREs. These studies reveal important direct chromatin effects, as well as limitations, of CFTR enhancers, and provide important considerations when targeting the CFTR locus for novel therapeutic gene editing.
2022-12-13 | GSE203558 | GEO
Project description:The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a TAD in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB Domain and CNC Homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (~8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.
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Project description:Looping of upstream cis-regulatory elements is required for CFTR expression in human airway epithelial cells [4C-seq]
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Project description:Three-dimensional genome structure plays an important role in gene regulation. Globally chromosomes are organized into active and inactive compartments, while at the gene level looping interactions connect promoters to regulatory elements. Topologically Associating Domains (TADs), typically several hundred kilobases in size form an intermediate level of organization. Major questions include how TADs are formed and what their relation is with looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb region on chromosome 7 surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Further, we find that these TAD boundaries are present irrespective of expression and looping of genes located between them. In contrast looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell type-specific long-range interactions and gene regulation. We investigated a 2.8 Mb region on Chromosome 7 (hg18 chr7: 115797757-118405450) containing the ENCODE region ENm001 42. The 5C experiment was designed to interrogate looping interactions between HindIII fragments containing transcription start sites (TSSs) and any other HindIII restriction fragment (distal fragments) in the target region. Libraries were generated for five cell lines: Caco2, Calu3, Capan1, GM12878 and HepG2, with two biological replicates for each line. 5C probes were designed at HindIII restriction sites (AAGCTT) using 5C primer design tools previously developed and made publicly available online at our My5C website (http://my5C.umassmed.edu). Probes were designed based on the ENCODE manual region 1 (ENM001) design 25 with additional probes placed throughout the region when appropriate. We also added probes to extend the analysis to include a 700 Kb gene desert region located directly adjacent to ENM001. Probe settings were: U-BLAST, 3; S-BLAST, 100; 15-MER, 3,000; MIN_FSIZE, 250; MAX_FSIZE, 20,000; OPT_TM, 65; OPT_PSIZE, 40. We designed 74 reverse 5C probes, and 605 forward 5C probes.
2016-01-07 | E-GEOD-75634 | biostudies-arrayexpress
Project description:The CFTR gene lies within an invariant topologically associated domain (TAD) demarcated by CTCF and cohesin, but shows cell-type specific control mechanisms utilizing different cis-regulatory elements (CRE) within the TAD. Within the respiratory epithelium, more than one cell type expresses CFTR and the molecular mechanism controlling its transcription are likely divergent between them. Here we determine how two extragenic CREs that are prominent in secretory epithelial cells in the lung, regulate expression of the gene. We showed earlier that these CREs, located at -44kb and -35 kb upstream of the promoter, have strong cell-type-selective enhancer function. They are also responsive to inflammatory mediators and to oxidative stress, consistent with a key role in CF lung disease. Here we use CRISPR/Cas9 technology to remove these CREs from the endogenous locus in human bronchial epithelial cells. Loss of either site extinguished CFTR expression and abolished long-range interactions between these sites and the gene promoter. The deletions also greatly reduced promoter interactions with the 5’ TAD boundary. We show substantial recruitment of RNAPII to the -35kb element and identify CEBPβ as a key activating transcription factor for airway expression of CFTR, likely through occupancy at both the CRE and the gene promoter.
2020-04-22 | GSE138166 | GEO
Project description:The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a TAD in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB Domain and CNC Homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (~8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.
2021-11-17 | GSE165718 | GEO